The goal of the proposed studies is to determine how retroviruses select and package their diploid RNA genomes. During the current funding period, we obtained strong in vivo and in vitro evidence that the Moloney Murine Leukemia Virus (MLV) uses an RNA structural switch mechanism, in which high affinity nucleocapsid (NC - the protein domain responsible for genome selection) binding sites are sequestered by base pairing in the monomeric RNA and become exposed to allow NC binding upon dimerization. Having nearly completed studies of this model retrovirus, we now intend to focus almost exclusively on the Human Immunodeficiency Virus (HIV-1). Methods developed over the past 3 1/2 years now enabled us to obtain NMR spectra of outstanding quality for the intact HIV-1 5'-UTR in its monomeric (356 nucleotides) and dimeric (712 nt) states. Based on these and other unpublished findings, we now believe that genome dimerization and packaging are mediated by a novel allosteric nucleotide displacement RNA switch mechanism, in which residues near the gag start codon induce a global rearrangement that simultaneously exposes a dimer-promoting stem loop (DIS) and high affinity NC binding sites that were sequestered in the momomeric conformer. Preliminary in vivo packaging experiments support this mechanism. 5'-UTR mutants that exclusively adopt either the monomer or dimer conformation have been prepared, and NMR spectra of outstanding quality have been collected for these constructs. We are thus poised to determine the high-resolution 3D structure of the HIV-1 5'-UTR in conformations relevant to the mechanism of diploid genome selection. NMR studies of such large RNAs are technically challenging - the average size of all NMR-derived RNA structures in RNA Structure Database is only 25 nucleotides - but the potential payoff is substantial, and could ultimately lead not only to a more detailed understanding of how HIV replicates, but also to the development of new approaches for the treatment of AIDS, cancers, and other virally-induced human diseases. PUBLIC HEALTH RELEVANCE: The aim of this project is to develop understanding of the mechanism that HIV-1 and other retroviruses use to select and package their RNA genomes. These studies should lead not only to a better understanding of how retroviruses replicate, but also to the development of new approaches for the treatment of AIDS, Cancer, and other virally-induced human diseases.